SK18102000A3 - Cell adhesion inhibitors and pharmaceutical composition containing them - Google Patents

Abstract

OMePUPA-V, (R)-N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-proly l-3-methyl)- beta -Alanine, a cell adhesion inhibitor, pharmaceutical compositions, and methods of treatment of cell-adhesion mediated pathologies.

Description

Technical field

The present invention relates to novel compounds useful for inhibiting, altering or preventing cell adhesion and cell adhesion related diseases. The invention also relates to pharmaceutical compositions comprising these compounds and methods of using them for inhibiting and preventing cell adhesion and cell adhesion related diseases. The compounds and pharmaceutical compositions of the invention may be used as therapeutic or prophylactic agents. They are particularly suitable for the treatment of many inflammatory and autoimmune diseases.

BACKGROUND OF THE INVENTION

Cell adhesion is the process by which cells are clustered together, migrated towards a specific target, or localized in the extracellular matrix. Cell adhesion alone is one of the underlying mechanisms involved in many biological phenomena. For example, cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and subsequent migration of these hematopoietic cells from the vessels and to the site of injury. Cell adhesion itself plays a role in many diseases, such as inflammatory and immune responses in mammals.

Investigations at the molecular level of cell adhesion have shown that various cell surface macromolecules, commonly known as cell adhesion molecules or receptors, mediate cell-cell and cell-matrix interactions. For example, proteins commonly referred to as integrins are a key mediator of adhesion interactions between hematopoietic cells and their microenvironment (Μ.Ξ. Hemler, VLA Proteins in the Integrin Family: Structures, Functions and Their Role on Leukocytes; Ann. Rev. Immunol. 8,

• • · • • · · · • • • e • · • • • • • · • · • • · • · • e • • · • • • • • · • • ···· • · • · • • · ·

p. 365 (1990)). Integrins are non-covalent heterodimeric complexes consisting of two subunits called α and β. There are 17 different α (α1-α, α-L, α-M, α-D, α-X, α-IIB, α-V and α-E subunits) and at least 9 different β (β1-β9) subunits that have been identified. Based on the type of components of the α and β subunits, each integrin molecule can be assigned to a subgroup.

Integrin α4-β1, also known as very late antigen-4 (VLA-4) or CD49d / CD29, is a leukocyte cell surface receptor involved in many different matrix adhesion interactions, both cell-cell and cell-matrix ( ME Hemler, Ann, Rev. Immunol., 8, p.365 (1990)). It serves as a receptor for cytokine-inducible surface protein endothelial cells, for vascular cell adhesion molecule-1 (VCAM-1), as well as for the extracellular matrix of fibronectin (FN) protein (Ruegg et al., J. Cell. Biol. 177, p. 179 (1991); Wayner et al., J. Cell Biol., 105, p. 1873 (1987); Kramer et al., J. Biol. Chem., 264, p. 4684 (1989) Gehlsen et al., Science, 24, 1228 (1988)). Anti-VLA-4 monoclonal antibodies (mAbs) have been shown to inhibit VLA-4-dependent adhesion interactions both in vitro and in vivo (Ferguson et al., Proc. Natl. Acad. Sci., 88, p. 8072 ( Ferguson et al., J. Immunol., 150, 1172 (1993)). Results of in vivo experiments suggest that this inhibition of VLA-4 dependent cell adhesion may prevent, inhibit or alter some inflammatory and autoimmune diseases (RL Lobb et al., The Pathophysiologic Role of α4 Integrins In Vivo, J. Clin, Invest., 94). , pp. 1722-28 (1994)).

In order to identify the minimum active amino acid sequences required for VLA-4 binding Komoriya et al. (The Minimal Essential Sequence for a Major Cell Type-Specific Adhesion Site (CS1) Within the Other Spliced Type III Connecting Segment of Fibronectin Is Leucine-Aspartic Acid-Valine, J.

• · ··

Biol. Chem. 266 (23), p. 15075-79 (1991)) synthesized a set of overlapping peptides based on the amino acid sequence of the CS-1 region (VLA-4 binding domain) of different fibronectin species. They identified an eight-amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, as well as two smaller overlapping peptides, Glu-Ile-Leu-Asp-Val and Leu-Asp-Val-Pro-Ser, having inhibitory activity against FN-dependent cellular adhesion. These results indicate that the tripeptide Leu-Asp-Val is the minimum sequence for cellular adhesion activity. Later, it was shown that Leu-Asp-Val binds only lymphocytes that show an activated form of VLA-4, and thus the question of the usefulness of these peptides in vivo arose (EA Wayner et al., Activation-Dependent Recognition by Hematopoietic Cells of LDV Sequence). in the Region of Fibronectin, J. Cell., Biol., 116 (2), pp. 489-497 (1992)). Certain larger LDV-containing peptides have been shown to in vivo activity (TA Ferguson et al., Two Integrin Binding Peptides Abrogate T-Cell-Mediated Immune Responses In Vivo, Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76 ( And SM Wahl et al., Synthetic Fibronectin Peptides Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and Recruitment, J. Clin Invest., 94, 655-62 (1994)). A cyclic pentapeptide has been described which can inhibit the adhesion of both VLA-4 and VLA-5 to FN (DM Nowlin et al., A Novel Cyclic Pentapeptide Inhibits α4β1 and α5β1 Integrin-mediated Cell Adhesion, J. Biol. Chem., 268 (27), pp. 20352-59 (1993) and PCT Application PCT / US91 / 04862). This pentapeptide is based on the Arg-Gly-Asp tripeptide sequence from FN, which is known as a common recognition site motif for several extracellular matrix proteins.

Examples of other VLA-4 inhibitors are disclosed, for example, in further US patent application 08 / 376,372 and in international patent application WO 98/04913, which are incorporated herein by reference. USSN 376,372 discloses linear β-amino acid containing peptidyl compounds having cellular adhesion inhibitory activity4. ______________________________________ < tb > ______________________________________ < tb > ··· ·· ········· International Patent Applications WO 94/15958 and WO 92/00995, incorporated herein by reference, disclose a cyclic peptide and peptidomimetic compounds having a modulating activity against cell adhesion. International patent applications WO 93/08823 and WO 92/08464 (incorporated by reference) disclose cell adhesion modulating compounds comprising a guanidyl group, a urea group and a thiourea group. U.S. Pat. No. 5,260,277 discloses cell adhesion modulating guanidinyl compounds (patent reference).

Despite these advances, specific inhibitors of VLA-4 dependent cell adhesion are required which have better pharmacokinetic and pharmacodynamic properties, such as oral bioavailability and long duration of action. These compounds could provide useful agents for treating, altering, preventing or suppressing various cell adhesion mediated diseases and VLA-4 binding.

SUMMARY OF THE INVENTION

The compounds of the present invention are VLA-4 integrin inhibitors and block the binding of VLA-4 to its various ligands, such as VCAM-1 and fibronectin regions. These compounds are useful for inhibiting the cell adhesion process, including cell activation, migration, proliferation and differentiation. The compounds are useful for inhibiting, preventing and suppressing VLA-4 mediated cellular adhesion and diseases associated with such adhesion, such as inflammatory and immune responses, including, for example, multiple sclerosis, asthma, allergic rhinitis, allergic conjunctivitis, inflammatory arthritis, autologous infection, rheumatoid arthritis. arthritis, organ, marrow transplant, septic restenosis inflammation, viral intestinal disease including disease, certain types of lung disease, type 1 diabetes, bone myocarditis transplantation, inflammatory ulcerative colitis and Crohn's toxic and immune nephritis, contact dermal hypersensitivity, psoriasis metastasis, psoriasis metastasis, psoriasis , multiple myeloma, and ate5

Φ • · • ···· • · · · · · • · • · • · • · • • · • • • · • • • • · • · • ···· • · · • •

rosklerózy. The compounds of the present invention may be used alone or in combination with other therapeutic or prophylactic agents to inhibit, alter, prevent or suppress cell adhesion. The present invention also provides pharmaceutical compositions comprising these VLA-4 mediated cell adhesion inhibitors and methods of using the compounds and compositions of the invention to inhibit cell adhesion.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: reports the sheep airway response after oMePUPA-V treatment. Sheep, naturally susceptible to Ascaris suum, are stimulated with an aerosol of Ascaris suum allergen, after 2 hours oMePUPA-V is administered at a labeled dose or equivalent amount of vehicle. At given times lung activity is measured and recorded as a change in specific airway resistance relative to baseline values prior to study (left panels). The airway resistance to inhaled carbachol is determined before the start of the study and 24 hours after allergen challenge (right panels). Respiratory response is recorded as the ratio of PC ₄ oo (amount of carbachol needed to increase resistance by 400%) of pre- and post-stimulation values.

Fig. 2: Sheep, naturally sensitive to Ascaris suum, are stimulated by administration of an oMePUPA-V aerosol at given doses or Ascaris suum. Changes in airway resistance are measured after aerosol challenge and the maximum lung resistance (cm H2 / sec) after challenge is compared to baseline values. * = p <0.05 compared to PBS control, single-factor analysis of variance followed by Dunnett's multiple comparison test with the control group. It indicates a statistically significant increase in the maximum specific lung resistance compared to the PBS control group.

Fig. 3: Sheep, naturally sensitive to Ascaris suum, are stimulated by administration of Ascaris suum aerosol 24 hours after a four-day administration of oMePUPA-V aerosol (0.03 mg) or an equivalent amount of vehicle (ethanol: normal saline, 1: 2) ···· ··· ·· ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

1: 499, lower top panel; Tris: normal panel). Lung activity is measured by change in line specific resistance before study (left panels). inhaled carbachol is recorded after allergen stimulation as a ratio of the increase in saline resistance at given times and recorded as the lung relative to baseline

Respiratory resistance is determined prior to inhalation study and 24 hours (right panels). PC ₄ oo Respiratory Response (amount of carbachol required to 400%) by comparing pre- and post-challenge values.

Fig. 4: Balb / c mice, pre-sensitized to DNFB, are stimulated by applying DNFB to the back of the left ear and vehicle to the back of the right ear. After 24 hours, the ear thickness is measured with a micrometer. oMePUPA-V is administered at the indicated doses 4 hours after DNFB stimulation. The positive control (+ CTRL) compound was administered at the maximum effective enteral dose. Values represent mean ± SD of 8 animals. The top panel shows the absolute swelling of the ear. The lower panel shows the percentage inhibition of ear swelling compared to vehicle-stimulated control (VEH).

Fig. 5: Analysis of competition between oMePUPA-V and known inhibitor under various activation conditions. Jurkat cells (1.5x10 ⁶ / ml) in TBS plus 2 mM Mn ²⁺ , 1 mM Ca ²⁺ plus 1 mM Mg ²⁺ , 1 mM Ca ²⁺ plus 10 mM Mg ²⁺ , 10 mM Mg ^2+, or 10 mM Mg ²⁺ plus 10 µM / π TS2 / 16 are treated with 5 nM ³ H known inhibitor alone or 5 nM ³ H known inhibitor plus 10 nM BIO1519 for 30 minutes at room temperature. The cells are then pelleted by centrifugation, resuspended in 100 μΐ TBS plus Mn ²⁺ and analyzed by scintillation counting. Binding numbers under these conditions indicate an integrin that is not occupied by the test compound and is therefore free to bind ³ H of a known inhibitor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds which are capable of forming a compound of formula (I). Inhibit VLA-4 mediated cellular adhesion by inhibiting ligand binding to this receptor. A preferred compound is (P) -N - [[4 - [[(2-methylphenylamino) carbonyl] amino] phenyl] acetyl] - which is referred to herein as oMePUPA-V.

prolyl-3-methyl-p-alanine,

oMePUPA-V

The present invention also relates to pharmaceutically acceptable derivatives, salts and esters of oMePUPA-V.

The compounds of formula I contain one or more asymmetric centers and can therefore exist in the form of racemic mixtures, individual enantiomers, diastereomeric mixtures and individual diastereomers. The present invention includes all such isomeric forms of the compounds of Formula I.

The present invention also includes pharmaceutically acceptable salts of the compounds of formula I. The term pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include ammonium, ammonium, calcium, copper, ferrous, ferric, Utne, magnesium salts, manganese, manganese, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts.

Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including natural substituted amines, cyclic amines, and basic ion exchangers.

• ···· • · · · ·· · • · • • • • · • • • · • · · · • • • • • • • • • • • • • • • · ···· • · · · ·

acids such as arginine, betaine, caffeine, choline, N, N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethenol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucamine, glucamine, glucamine, glucamine, glucamine, glucamine, glucamine, methylglucamine, polyamine resins, procaine, histidine, hydrabamine, isopropylmorpholine, piperazine, piperidine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, trometamine and the like.

If the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid and the like. Particularly preferred are citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid and tartaric acid.

Further, the present invention includes prodrugs, especially ester prodrugs, wherein the (R) -N - [[4 - [[(2-methylphenylamino) carbonyl] amino] phenyl] acetyl] -L-propyl-3-methyl-p-alanine carboxyl group is esterified with any alcohol. Preferred alcohols include methanol, ethanol, propanol, butanol or C 1 -C 10 alcohols having straight or branched chain.

The ability of the compounds of formula I to antagonize the action of VLA-4 makes them suitable for preventing, treating or reversing the symptoms, disorders or diseases caused by the binding of VLA-4 to its ligands. Thus, these antagonists will inhibit the processes

Cell adhesion, including cell activation, migration, proliferation and differentiation. A further object of the present invention is a method of treating, preventing, ameliorating or suppressing diseases or disorders mediated by the VLA-4 pathway. Such diseases include, for example, asthma, multiple sclerosis, allergic rhinitis, allergic conjunctivitis, inflammatory pulmonary disease, rheumatoid arthritis, multiple myeloma, septic arthritis, type I diabetes, organ transplant rejection, inflammatory bowel disease, and others.

The compounds of the present invention can be synthesized using conventional procedures, some of which are exemplified herein. These compounds are preferably chemically synthesized from readily available starting materials such as α-amino acids and functional equivalents thereof. Modular and convergent methods for the synthesis of these compounds are also preferred. For example, in a convergent approach, large portions of the resulting product are combined in the last steps of the synthesis, rather than gradually adding small portions to the growing chain of the molecule.

The compounds of the present invention may also be modified by attaching suitable functional groups to improve their selective biological properties. These modifications are known to those skilled in the art and include modifications that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral bioavailability, increase solubility to allow them to be injected, altering metabolism and varying the rate of elimination. Examples of such modifications include, but are not limited to, esterification with polyethylene glycols, derivatization with pivolate or fatty acids, conversion to carbamates, hydroxylation of aromatic rings, and substitution of aromatic ring heteroatoms.

According to the present invention, the term "patient" means "mammalian". · ··· ··· ·· · · ·· ·· · ca, including man. The term cell means any cell, preferably a mammalian cell, including a human cell.

The activity and specificity of the prepared compounds of the present invention with respect to VLA-4 can be determined by in vivo and in vitro assays.

For example, the cell adhesion inhibitory activity of the compounds of the present invention can be measured by determining the concentration of inhibitor required to block binding of VLA-4 expressing cells to fibronectin or CS1 coated microtiter plates. In this type of assay, wells of a microtiter plate are coated with either fibronectin (containing the CS-1 sequence) or CS-1. If CS-1 is used, it must be conjugated to a protein carrier such as calf serum albumin to bind to the wells. When wells are coated, various concentrations of test compound are added along with appropriately labeled cells expressing VLA-4. Alternatively, the test compound may be added first and allowed to incubate with the coated wells prior to the addition of the cells. The cells are incubated in the wells for at least 30 minutes. After incubation, the wells are emptied and washed. Inhibition of binding is measured by determining the value of plate-bound fluorescence or radioactivity at each different concentration of test compound, as well as a control sample not containing test compounds.

VLA-4-expressing cells that can be used in this assay include Ramos cells, Jurkat cells, A375 melanoma cells, as well as human peripheral blood lymphocyte (PBL) cells. The cells used in this assay can be labeled by any suitable method, for example, using fluorescent or radioactive labels.

A direct binding assay may also be used to quantitate the inhibitory activity of the compounds of the invention. In this assay, a VCAM-IgG fusion protein comprising the first two immunoglobulin domains of VCAM (D1D2) linked above the core region of the IgG1 molecule (VCAM 2D-IgG) is conjugated to a labeling enzyme such as alkaline phosphatase (AP). The synthesis of this VCAM-IgG fusion is described in International Patent Application WO 90/13300, the contents of which are incorporated herein by reference. The conjugation of this fusion to the labeling enzyme is achieved by cross-linking procedures known to those skilled in the art.

The VCAM-IgG enzyme conjugate is then placed in the wells of a multi-well microtiter plate, such as a plate that is part of the Millipore Multiscreen Assay System (Millipore Corp., Bedford, MA). Thereafter, different concentrations of test inhibitor compound are added to each well followed by cells expressing VLA-4. The cells, compound and VCAM-IgG enzyme conjugate are mixed and allowed to incubate at room temperature.

After incubation, the wells are dewatered under vacuum and the cells and all bound VCAM remain in the wells. The amount of bound VCAM is determined by adding a suitable colorimetric substrate for the VCAM-IgG enzyme conjugate and determining the amount of reaction product. A decrease in the amount of reaction product means an increase in binding inhibitory activity. The procedure for some tests is described below.

To test the inhibitory specificity of the compounds of the present invention to VLA-4, assays are performed for major groups of integrins, i.e., β2 and β3, as well as for other βΐ integrins such as VLA-5, VLA-6 and α4β7. These assays may be similar to the adhesion and direct binding inhibition assays described above, replacing the respective integrin-expressing cells and the corresponding ligand. For example, polymorphonuclear cells (PMN) expressing β2 integrins and ICAM binding on their surface are used. Β3 integrins are involved in platelet aggregation and inhibition can be measured using a standard platelet clotting assay. VLA-5 binds specifically to Arg-Gly-Asp sequences, while VLA-5 binds to laminin. α4β7 is a recently discovered homologue of VLA-4, which also binds fibronectin, which also binds fibronectin. · ··· ·· ···· ·· · and VCAM. Α4β7 specificity is determined in a binding assay using the VCAM-IgG-enzyme-labeled conjugate described above and a cell strain that expresses α4β7 but not VLA-4, such as RPMI-8866 or JY cells.

Once VLA-4 specific inhibitors have been identified, they can be further characterized by in vivo assays. One such assay studies the inhibition of contact hypersensitivity in animals and is described, for example, in P.L. Chisholm et al., Monoclonal Antibodies to the Integrin α-4 Subunit Inhibit the Murine Contact Hypersensitivity Response, Eur. J. Immunol., 23, 682-688 (1993) and in Current Protocols in Immunology, J.E. Coligan et al., Eds., John Wiley &amp; Sons, New York, 1, p. 4.2.1-4.2.5 (1991), the contents of which are incorporated herein by reference. In this test, the animal skin is sensitized by exposure to an irritant such as dinitrofluorobenzene, then easily physically irritated, for example, by lightly scratching the skin with a sharp edge. After recovery, the animals are sensitized again following the same procedure. Several days after sensitization, one ear of the animal is exposed to a chemical vehicle and the other ear is treated with a non-irritating control solution. Shortly after ear irritation, animals are injected subcutaneously with various doses of the VLA-4 inhibitor. In vivo inhibition of cell adhesion-related inflammation is tested by measuring the swelling response of the treated ear against the swelling of the untreated ear of the animal. Swelling is measured with a caliper or other instrument suitable for measuring ear thickness. In this way it is possible to determine which inhibitors of the present invention are most suitable for inhibiting inflammation.

Another in vivo assay that can be used to test the inhibitors of the invention is the sheep asthma assay. This assay is performed according to the procedure described in W.M. Abraham et al., Α-Integrins Mediated Antigen-Induced Late Bronchial Responses and Prolongated Airway Hyperresponsiveness in Sheep, J. Clin. Invest., 93, p. 776-87 (1994), which is referred to herein as 9 9-9 (1994). 9

999 999 99 9999 99 · Ref. This assay measures the inhibition of the late phase airway response and Ascaris-induced airway hypersensitivity in allergic sheep. The compounds of the invention may also be tested in a platelet aggregation assay.

Surprisingly, the VLA-4 inhibitors of the present invention are preferably active and selective. Generally, these compounds are selective for VLA-4 (> 10000 fold over α4β7 and α5β1), are negative in conventional PanLabs and non-GLP Arnes assays, pure in standard pharmacological side-effect tests, and are effective in a sheep model of predictive amounts effective for humans. 1 mg / day or less, once daily.

Surprisingly, the compounds of the invention have good activity compared to structurally similar VLA-4 inhibitors. For example, in Ascaris susceptible sheep treated once daily for four days with a nebulized drug at a dose of 0.1 mg / kg and then challenged with antigen 24 hours after the last dose, previously tested compounds significantly reduced early response and blocked later phase bronchoconstriction and development of non-specific hypersensitivity. Assuming biological equivalence in humans, the total dose for humans weighing 70 kg could be 7 mg. Further, the drug administered to sheep via the endotracheal tube at given doses is estimated to be twice as high as typically achieved in humans by oral inhalation device. Further, it is likely that additives will need to be added to the resulting solid composition in order to optimize device loading and drug delivery. Based on these factors, a human dose of 14 mg or more can be estimated, which exceeds the technical limit of 1 to 5 mg, which can be delivered in a single dose using a dry powder inhaler (DPI). Although it is possible to deliver the required dose with several doses of DPI, this will present a disadvantage in the asthma medication market, where the typical dose of inhaled steroid is 0.2 to 1.0 mg.

oMePUPA-V reduced early response, blocked late phase bron14 · ···· ········································· And normalized hypersensitivity at a minimum dose of 0.003 mg / kg when administered as a spray dose 2 hours prior to antigen challenge. Furthermore, a daily dose of 0.001 mg / kg for 4 days with antigen challenge 24 hours after the last dose gave a maximal response. oMePUPA-V is 30 to 100 times more potent than previously used compounds at dosing levels in the range that is the same as for the best marketed steroids for inhalation use. oMePUPA-V, as well as the penultimate synthetic intermediate, is highly crystalline (see Figure 1, Table 1).

Furthermore, oMePUPA-V has an improved metabolic profile compared to known VLA-4 inhibiting compounds. For example, after aerosol administration, the claimed compounds rapidly convert to a less active metabolite, which is the predominant product derived from bronchoalveolar lavage (BALF) and is also the predominant product observed in systemic circulation, exhibiting a longer plasma half-life than the parent compound. Since rapid metabolic conversion to less active compounds is a suitable strategy to reduce system exposure, compounds that metabolize to by-products with little or no intrinsic activity present less developmental difficulties and are beneficial in accumulation.

Potential proteolytic products of oMePUPA-V are inactive in the VLA-4 binding assay and thus inactive products will be generated by proteolysis. However, in vitro metabolic studies as well as in vivo PK studies in rats, dogs and sheep show that oMePUPA-V is proteolytically stable.

Table 1:

Properties of oMePUPA-V compared to known inhibitors

property Known compound oMePUPA-V IC ₅₀ against VLA-4-VCAM-Ig binding 1.5 nM 2.7 nM IC ₅₀ against α4β7-VCAM-Ig binding 2.7 μΜ > 100 μΜ _IC50 of adhesion to FN-a5Pl > 100 μΜ > 100 μΜ

· 99 99 99 9

9 9 9 9 9

999 9 9 9 9 9 9

9 9 9 9 9

9 9 9 9 9

999 99,999 99,999

Minimum effective dose in sheepmeat deli - single dose 0.1 mg / kg 0.003 mg / kg - repeated dose 0.03-0.1 mg / kg 0.001 mg / kg crystallinity not Yes In vitro pharma screen 85 test 1 weak hit clean ACE activity ACE substrate clean glucuronidation low levels planned Ames test (nutagenicity) clean clean Ancillar Pharmacology (side effects) clean Systemic exposure, dose of 10 mg aerosol (sheep) 40 ng / mlxh. Finished Metabolic profile active metabolite Oral bioavailability <1% <1% Large stability (40 ° C, 75% relative humidity) unstable stable> 4 weeks Stability in formulation (40 ° C) 0.2% degradation / day stable> 4 weeks Compounds of the invention can be modified into pharmaceuticals

compositions which can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted container. The term parenteral administration includes subcutaneous, intravenous, intramuscular, intra-articular, joint, intrasternal, intrathecal, intra-hepatic, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions of the invention comprise any compound of the invention or a pharmaceutically acceptable derivative thereof, together with any pharmaceutically acceptable carrier. The term carrier includes pharmaceutically acceptable adjuvants and vehicles. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the invention include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, partially glyceridated mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulphate, dibasic phosphate, am · · · · · · · · · · · Sodium, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and lanolin.

According to the invention, the pharmaceutical compositions may be in the form of a sterile injectable solution, for example, a sterile aqueous injectable solution or an oily suspension for injection. Such suspensions may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable compositions may be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, stabilized oils are employed as a solvent or suspending agent. For this purpose any bland fixed oil may be employed including mono- and diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables as well as pharmaceutically acceptable natural oils, such as olive oil or castor oil, especially in their polyoxyethylated forms. These oily solutions or suspensions may also contain long-chain alcohols as a diluent or dispersant.

The pharmaceutical compositions of the invention may be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral administration, lactose and corn starch are commonly used as carriers. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, suitable diluents are lactose and dried corn starch. If he's on oral

• ···· • · · · · · • • · • • • • · • · • · • · · · • • • • • • • • • • • • • • · · ···· · · · · ·

using the required aqueous suspension, the active ingredient is mixed with an emulsifier and a suspending agent. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of the invention may be administered in the form of suppositories for rectal administration. Suppositories may be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and melted in the rectum to release the drug. Such materials are cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the present invention can be administered topically, especially if the site of treatment is sites or organs that are readily accessible for topical application. These include eye disease, skin disease or rectal disease. Suitable pharmaceutical formulations are readily prepared for each of these areas or organs.

Topical administration to the lower intestinal tract may be by rectal suppository form (see above) or by an appropriate enema formulation. Topical transdermal patches may also be used.

For topical application, the pharmaceutical compositions of the present invention may be formulated in suitable ointments containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oils, liquid petroleum petrolatum, white petroleum petrolatum, propylene glycol, polyoxyethylene compounds, emulsifying waxes, and water. Alternatively, the pharmaceutical compositions may be prepared in a suitable solution or cream containing the active ingredients suspended or dissolved in one or more pharmaceutical carriers. Suitable carriers include mineral oil, sorbitan monostearate, poly18, polyol, sorbitan monostearate, poly18. Sorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water, but the present invention is not limited to these examples.

For ophthalmic use, the pharmaceutical compositions may be prepared as micronized suspensions in isotonic, pH adjusted, sterile saline, or preferably as solutions in isotonic, pH adjusted, sterile saline, either with or without preservatives such as benzylalkonium chloride. Alternatively, the ophthalmic pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions of the present invention may also be administered by nasal aerosol or by inhalation using a nebulizer, a dry powder form for inhalation or a metered dose inhaler. Such compositions are prepared in accordance with techniques known to those skilled in the art of pharmaceutical compositions and may be prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons and / or other conventional solubility enhancers or dispersants. Further, the compositions of the invention may contain any pharmaceutically acceptable carriers, such as lactose for dry powder compositions.

The amount of active ingredient that may be combined with the carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Obviously, the specific dose and treatment regimen for a particular patient will depend on various factors including compound activity, age, body weight, general health, sex, diet, time of administration, elimination rate, drug combination, and physician judgment and severity of the disease. that is being treated. The amount of active ingredient may also depend on the therapeutic or prophylactic agent with which the ingredient is used. · ··· ··· ··· ·· ···· ·· ··· Serves.

The dosage and dose interval of the compounds of the present invention effective in preventing, suppressing or inhibiting cell adhesion will depend on various factors such as the nature of the inhibitor, the size of the patient, the target of treatment, the nature of the disease being treated, the particular pharmaceutical composition used, and the judgment of the physician. . Dosage levels in the range of 0.001 to 100 mg / kg body weight per day, preferably in the range of 0.01 to 50 mg / kg body weight per day, and more preferably about 10 mg / kg body weight per day, of the active ingredient of the compound .

For use as a composition for intravenous administration, a dosage of 0.001 mg to 25 mg / kg, more preferably about 0.01 mg / kg to 1 mg / kg is suitable.

In accordance with another embodiment, compositions comprising a compound of the present invention may also contain other agents selected from the group consisting of corticosteroids, bronchodilators, antiasthmatics (cell stabilizers), anti-inflammatory agents, antirheumatics, immunosuppressants, antimetabolites, immunomodulators, antipsoriatics and anatidiabetics. Specific compounds of these classes may be selected from those listed under the appropriate name in Comprehensive Medicinal Chemistry, Pergamon Press, Oxford, England, p. 970-986 (1991), which is incorporated herein by reference. In addition to this group, the compositions may include compounds such as theophylline, sulfasalazine, and aminosalicylates (anti-inflammatory agents); cyclosporin, FK-506, and rapamycin (an immunosuppressant); cyclophosphamide and methotrexate (antimetabolics); steroids (by inhalation, oral and topical administration) and interferons (immunomodulators). Further, the compounds of the present invention may be administered together with other cell adhesion inhibitors. When one or more additional agents are administered in combination with the claimed VLA-4 inhibitors, the active ingredients may be formulated together or alternatively may be administered in combination. Serving venom20 · · · 20 jed 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 The active or multiple active agents in combination with the VLA-4 inhibitors of the invention may be substantially simultaneous or sequential. One skilled in the art can readily determine the most appropriate route of administration depending on the agent to be administered, the desired outcome and the patient and the disease to be treated.

In accordance with other embodiments, the present invention provides methods for preventing, inhibiting, or suppressing inflammation associated with cell adhesion and immune or autoimmune responses associated with cell adhesion. Cell adhesion associated with VLA-4 plays a key role in a variety of inflammations, immune and autoimmune diseases. Inhibition of cell adhesion by the compounds of the present invention can be used in methods of treating or preventing inflammatory, immune or autoimmune diseases. Preferably, the diseases to be treated by the methods of the present invention are selected from the group consisting of asthma, arthritis, allergies, adult respiratory stress syndrome, cardiovascular disease, thrombosis or harmful platelet aggregation, transplant rejection, neoplastic diseases, psoriasis, sclerosis. multiplex, diabetes and inflammatory bowel disease.

These methods may utilize the compounds of the present invention in monotherapy or in conjunction with anti-inflammatory or immunosuppressive agents. Such combination treatment comprises administering the agent in a single dosage form or in multiple dosage forms administered at the same time or at different times.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of oMePUPA-V oMePUPA-V, which is (R) -N - [[4 - [[(2-methylphenylamino) carbonyl] ·····

· 4 · 4 · 4 · 9 · 9 · 9 · 9

9999 99999 amino] phenyl] acetyl] -L-prolyl-3-methyl-p-alanine, prepared by sequential synthesis from commercially produced Boe- (L) -proline (Boc-Pro-OSu; Bachem) and hemisulfate (R) benzyl 3-aminobutyrate (Celgene Corp.). The starting materials are condensed in dichloromethane in the presence of triethylamine, then the 4 N hydrochloric acid in dioxane is hydrolyzed to give the hydrochloride which is recrystallized from a mixture of dichloromethane and diethyl ether. By condensation of the hydrochloride with succinimidyl-2- [4- [2- (methylphenylaminocarbonyl)] aminophenylacetate (MPUPA-OSu), prepared from the corresponding acid, MPUPA-OH (Ricerca, Inc.), a crystalline oMePUPA-V-benzyl ester is obtained which The reaction mixture was subjected to catalytic hydrogenation (10% palladium on carbon) in tetrahydrofuran / water (9: 1) to give oMePUPA-V. The resulting product was obtained as a white solid after recrystallization from 20% aqueous acetone.

Summary of physical properties:

Chemical name: (R) -N - [[4 - [[(2-methylphenylamino) carbonyl] amino] phenyl] acetyl] -L-prolyl-3-methyl-4-alanine,

General formula: C25H30N4O5 Molar mass: 466.53 Appearance: pure white powder Melting point: 153.6-154.4 ° C.

Scheme 1:

Preparation of MPUPA-OS (2) from MPUPA-OH (1)

- ^0C, * ^CH3CN ·· Ql _n a _n ^{CTt 0SU} _ru O n 2. HOSu, TEA, -60 °, 0.5 h IMH ³ 1 then tt, 2 hr. CH ₃ 2

Scheme 2:

Synthesis of oMePUPA-V (8) from Boe- (L) -Pro-OSu (3) and Benzyl (R) -3-Aminobutyrate Hemisulfate (4)

• • · • ···· • · · · • • · • · • · • · · ·· • · • • • · • · • · • · · • • · • • • ·· · • ··· • · ·· • ···· • · ·· ·· ·

N HCl / dioxane

rt .. 2 h

92% (2 steps)

H ₂ /10% Pd, 10% aq. THF

380 kPa, mp, 25 h., 87% ² · recrystallization from m.p.

20% aq. acetone ⁸⁴ *

Synthesis of oMePUPA-V

The synthetic procedure used to prepare oMePUPA-V is described in Schemes 1 and 2. Starting materials are obtained from commercially available compounds: Compound (1) is prepared in large quantities by Ricerca, Inc., Painesville, OH; compound (3) is obtained from Bachem Bioscience, Inc., King of Prusia, PA and compound (4) from Celgene Corp., Warren, NJ.

Preparation of oMePUPA-V:

General analytical methods ^(X H NMR, ¹³ C NMR, MS, IR &^{HPLC); H} NMR were run either on a Bruker AC 300 or a Varian 500 or a Varian 600 instrument and samples were analyzed either on perdeuterodime sulfoxide and referenced to DMSO (δ

2.49 ppm) or in deuterochloroform and refer to the remainder

• · • · · • · • · • · • • · • • • ♦ · • • • · • ···· · · • · ·

• ···········

• Chloroform (δ 7.24 ppm).

¹³ C NMR is measured on either a Varian 500 or Varian 600 instrument and the samples are measured either in perdeuterodimethylsulfoxide and refer to perdeuterodimethylsulfoxide (δ 40.5 ppm) or deuterochloroform and refer to deuterochloroform (δ 77.0 ppm).

Mass spectra are measured on a VG Platform LC-MS-DS Mass Spectrometer Hewlett Packard Model 1500 AutoSampler System and the data is processed using a Fisons VG MassLynx Mass Spectrometer Workstation. HRMS is performed on M-Scan (PA) using fast atom bombardment (FAB) on a VG Analytical ZAB 2SE mass spectrometer with respect to SOP # MS-002, MS-006, MS-012 and MS-023. Cesium ion bombardment is used to generate the mass spectrum ions recorded by the PDP-11-250J.

IR spectra are measured on a Perkin Elmer 1600 Steri FTIR instrument.

Analytical high pressure chromatography (HPLC) is performed as follows:

1. Chromatograms using program 1 (Equilibrate @ 20% B, sample injection, 20% B (1 min), 20% to 70% B (24 min), 70% -100% B (70 min) with an Perkin Elmer 785A UV detector (set at 214 nm) and an Applied Biosystems 783A UV detector (set at 254 nm) with a PE Nelson 1020 integrator.

2. Chromatograms using program 8 (Equilibrate @ 15% B, Sample Injection, 15% B (1 min), 15% -40% B (25 min), 40% B (10 min) are obtained with Applied Biosystems 400 Solvent Delivery System at 783 A wavelength on a UV detector using a Waters 717 autosampler The data is processed using the Hew • lett Packard 3396 Sater II integrator. The integrator is set to the following parameters: damping = 8, baseline = 5, rejected area = 10000, peak width = 0.04, recording speed = 2.

Each HPLC is performed using a Vydac C-18 column (pore size 5 µ, 4.5 mm x 25 cm, cat # 218TP54).

Solvent A (water + 0.1% trifluoroacetic acid)

Solvent B (acetonitrile + 0.1% trifluoroacetic acid)

Flow rate = 1 ml / min.

The following gradients are used:

Program 1: Equilibrate @ 20% B, sample injection, 20% B (2 min), 20% to 70% B (25 min), 100% B (5 min).

Physical data for [4 - [[[(2-methylphenyl) amino] carbonyl] amino] phenyl] acetic acid (MPUPA-OH: manufactured by Ricerca Inc.):

mp 210-215 ° C (dec.);

IR (KBr) 3295 (broad band), 3034 (broad band), 1707, 1637, 1603, 1551, 1516, 1457, 1414, 1302, 1241, 1189, 1118 cm ^-1 ;

NMR (600 MHz, CDCl 3) δ 12.28 (bs, 1H), 9.0 (s, 1H), 7.91 (s, 1H), 7.88 (d, J = 7.8Hz, 1H), 7.43 (d, J = 8.4 Hz,

2H), 7.19 (d, J = 8.4 Hz, 2H), 7.16 (m, 2H), 6.94 (dd, J = 7.8,

8.4Hz, 1H), 3.51 (s, 2H), 2.25 (s, 3H);

¹³ C NMR (150 MHz, perdeuterodimethylsulfoxide) δ 173.0 (C), 152.7 (C), 138.5 (C), 137.5 (C), 130.2 (CH), 129.8 (CH) ), 128.3 (CH),

127.5 (CH), 126.2 (CH), 122.7 (CH), 121.0 (CH), 118.1 (CH), 40.1 _(CH2), 17.9 (CH ₃₎ ;

MS (EI) m / z 285 (M + 1) ⁺ 193, 152, 134, 132, 109, 108, 106, 93, 91, 57;

Elemental analysis for C ₁₆ H ₁₆ N ₂ O ₃ : Calculated C 67.59; H, 5.67; N, 9.85; Found: C, 67.60; H, 5.70; N, 10.01.

Preparation of succinimidyl [4 - [[[(2-methylphenyl) amino] carbonyl] amino] -

Phenyl acetate acetate (2, MPUPA-OSu) • · · · · · · · · · · · · · · · · · · · · ·

To a boiling suspension of 150 g of o-methylphenylureaphenylacetic acid (1, MPUPA-OH; 0.501 mol; from Ricerc, Inc.) in 600 ml of acetonitrile (600 ml) was added thionyl chloride (41 ml, 0.558 mol) over 10 minutes with vigorous stirring. A large amount of hydrogen chloride is released. The reaction mixture was stirred with stirring

Cool to room temperature for 1.5 hours. The reaction mixture turned into a pink suspension, to which 75.5 g (0.636 mol) of N-hydroxysuccinimide (HOSu) was added in one portion. 174 ml of triethylamine are added dropwise over 30 minutes at a temperature which is kept below 60 [deg.] C. with a water bath. Stirring is continued for 2 hours and then distilled water is added to the reaction mixture. The solid is filtered off and washed with 2 liters of distilled water and twice with 200 ml of acetonitrile, air dried and then dried over P2O5 under vacuum (about 13.3 Pa) to give 175 g (yield 97%) of the crude product as a beige powder. 174 g of crude product were recrystallized from 3.5 liters of acetonitrile with 10 g of activated carbon to give 129 g of MPUPA-OSu (2; yield 68%) as a white powder (purity &gt; 99%).

Melting point: 211.2-211.8 ° C;

IR (KBr): 3905-3203 (broad band), 1816, 1783, 1654, 1368, 1304, 1244, 1116, 1021 cm ^-1 ;

^{Χ Η} NMR (300 MHz, _DMSO-d6): δ 9.04 (s, 1H), 7.92 (s, 1H), 7.82 (d, 1H), 7.44 (d, J = 8 5Hz, 2H), 7.24 (d, J = 8.5Hz, 2H), 7.15 (m, 2H), 6.93 (dd, J = 7.4, 7.3Hz, 1H), 4 0.02 (s, 2H), 2.80 (s, 4H), 2.23 (s, 3H);

MS (EI, ES ⁺ ) m / z 382 [(M + 1) &lt; ^{+ &gt};], 239, 108, 106.

Physical data for succinimidyl Boe (L) -proline (Boc-Pro-OSu; 3; substance obtained from Bachem Bioscience):

M.p .: 132-136 ° C;

IR (KBr) 3456, 2940, 1731, 1619, 1561, 1541, 1497, 1454, 1395,

1337, 1259, 1202, 1118, 1060 cm ^{@ -1} ;

...... ·? ·? ·?

......;

• · · · · · · ·

J = 3.8, 8.7Hz,

2.32 (m, 1H);

^{······; H} NMR (300 MHz, CDCl) δ 4.51 (dd, 1H), 3.56 (m, 1H), 3.44 (m, 1H), 2.80 (s, 4 H )

2.27 (m, 1H); 1.94 (m, 2H); 1.43 (s, 9H);

MS (EI) m / z 335 (M + N ₂ ) ⁺ 279, 213, 138, 114, 86;

HPLC 97.1%,

Physical data for benzyl (R) -3-aminobutyrate hemisulphate (4; substance obtained from Celgene Corp.):

mp 249.4-249.8 ° C;

IR (KBr) 3515, 3383, 2989, 2945, 2880, 1821, 1788, 1744, 1701,

1476, 1454, 1421, 1394, 1368, 1260, 1241, 1202, 1159, 1077 cm ^-1 ;

NMR (300 MHz, CDCl3) δ 7.85 (bs, 2H), 7.26 (s,

5H), 5.06 (ABkv, J = 12.3Hz, 2H), 4.35 (m, 2H), 3.73 (m, 1H), 2.92 (dd, J = 6.4, 17, 1Hz, 1H), 2.66 (dd, J = 6.4, 17.0Hz, 1H), 1.35 (d, J = 6.5Hz, 3H);

MS (EI) m / z 195 (M + 3) ^&lt; ^{+ &gt};, 194 (M + 2) ^&lt; ^{+ &gt};, 106, 92, 91;

HPLC 99.0%.

Preparation of N- (tert-butoxycarbonyl) -L-prolyl-3-methyl- (R) -β-alanine benzyl ester (5)

To a well-stirred suspension of 66.7 g of benzyl R-3-aminobutyrate hemisulfate (4; 213 mmol) in 200 mL of dichloromethane was added

53.9 g of Boe- (L) -Pro-OSu (3; 222 mmol) and 95 ml (681 mol) of triethylamine. The reaction mixture was allowed to stir at room temperature for 2 hours. The reaction mixture is partitioned between 1.5 liters of ethyl acetate and 250 ml of water and the organic layer is washed three times with 250 ml of 10% citric acid, 250 ml of water, 250 ml of saturated sodium bicarbonate solution, 250 ml of water and three times with 250 ml of brine. sodium sulfate and evaporated first by rotary evaporation (40 ° C, 10.7 kPa) and then under high vacuum (room temperature, 16 hours; 26.7 Pa) to give 88.1 g of intermediate 5 as a viscous oil which contains residues of ethyl acetate and dichloromethane (by NMR) and has a purity greater than ·· ··

98% (HPLC). This material was used in the next reaction without purification.

^X H NMR (300 MHz, CDCl) δ 7.30 (m, 5 H), 6.44 (bs,

1H), 5.10 (dd, J = 12.3, 14.1Hz, 2H), 4.32 (m, 1H), 4.13 (m, 1H), 3.34 (bs, 2H), 2 48 (d, J = 5.1 Hz, 2H), 2.1 (m, 2H), 1.75 (bs, 2H), 1.40 (s, 9H), 1.17 (d, J = 6) 0.5 Hz, 3H);

MS (EI): m / z [M + Na] ^&lt; ^{+ &gt;} 313, 291, 191, 194, 165, 91.

Preparation of L-prolyl-3-methyl- (R) -β-alanine benzyl ester hydrochloride (6)

To intermediate 5 from the previous reaction was gradually added 240 mL of a 4N hydrochloric acid in dioxane solution. Strong gas evolution (exothermic). The reaction mixture was allowed to stir for 2 hours at room temperature and then evaporated first on a rotary evaporator (45 ° C, 10.7 kPa) and then overnight under high vacuum (room temperature, 14 hours, 26.7 Pa) to give ultra-viscous, crystallized from dichloromethane / ether (600 mL / 700 mL) to give 64.0 g (92% overall yield over 2 steps) of the hydrochloride 6 as a white solid (HPLC purity 99.6%) .

Melting point: 119.8-120.5 ° C

IR (KBr): 3217, 3072, 2904, 2756, 1736, 1681, 1560, 1446, 1387, 1352, 1295, 1244, 1178, 1096 cm ^-1 ;

¹ H NMR (500 MHz, CDCl 3) δ 10.21 (bs, 1H), 8.71 (d,

J = 8.0Hz, 1H), 7.77 (bs, 1H), 7.24 (m, 5H), 5.00 (s, 2H), 4.52 (bs, 1H), 4.22 (t J = 6.5Hz, 1H), 3.33 (bs, 2H), 2.67 (dd, J = 5.5, 15.5Hz, IH), 2.44 (m, 2H), 1.89 (m, 3H), 1.15 (d, J = 6.5 Hz, 3H);

¹³ C NMR (125 MHz, CDCl 3) δ 171.03 (C = O), 167.67 (C = O), 135.58 (C), 128.43 (CH), 128.13 (CH), 128 , 06 (CH), 66.34 (CH ₂ ), 59.71 (CH), 46, 55 (CH ₂ ), 43, 34 (CH), 40.42 (CH ₂ ), 30.50 (CH ₂₎ 24.23 (CH ₂ ), 19.92 (CH ₃ );

MS (EI) m / z 291 [M-Cl] ^&lt; ^{+ &gt;} 199, 194, 160, 139, 92, 91;

H, N ₃ CieHz O3Cl _2: Calculated C 58.80; H, 7.094;

····

N, 8.57; Found: C, 58.95; H, 6.99; N, 8.46.

Preparation of N- [[4 - [[(2-methylphenylamino) carbonyl] amino] phenyl] acetyl] -L-prolyl-3-methyl- (R) -β-alanine benzyl ester (7)

To a solution of 61.77 g of hydrochloride 6 (189 mmol) in 125 ml of DMF was added 69.39 g of MPUPA-OSu (2; 181.9 mmol) and then 90 ml of triethylamine (pH 10). The reaction mixture was allowed to stir for 3.5 hours and then diluted with 1 L of ethyl acetate and extracted three times with 250 mL of water. The product now begins to precipitate. 250 ml of a 10% citric acid solution (exothermic) are added to the organic layer and a large amount of precipitate is formed upon shaking. Filter the solid on a frit (2 L, M). The solid is washed twice with 250 ml of 10% citric acid, 250 ml of water, twice with 250 ml of saturated sodium bicarbonate solution, 250 ml of water and three times with 250 ml of brine and then dried on a funnel (about 10.7 kPa) overnight ( 14 hours) to give an off-white solid which was recrystallized from tetrahydrofuran / diethyl ether (1 liter / 1.4 liter) to give 83.3 g of compound 7 (HPLC purity 99.6%) as a white solid. .

The filtrate is then washed three times with 250 ml of 10% citric acid, 250 ml of water, twice with 250 ml of saturated sodium bicarbonate solution, 250 ml of water and three times with 250 ml of brine. Each additional wash with an aqueous solution precipitates another compound; washing was continued and care was taken not to lose the precipitate. After filtration, 4.02 g of product is obtained as a white solid. The filtrate was finally diluted with 1 L of ether, filtered and washed three times with 100 mL of diethyl ether to give an additional 1.67 g of a white solid. The overall yield of this reaction was 88%.

Melting point: 153-153.5 ° C

IR (KBr) 3342, 3307, 3119, 2966, 1737, 1702, 1643, 1590, 1543,

1514, 1455, 1414, 1308, 1238, 1179 cm ^-1 ;

¹ H NMR (500 MHz, DMSO-d6): 3: 2 rotamers mixture (predominant conformation peaks): δ 9.00 (bs, 1H), 7.91 (bs, 1H), 7.84 (d, J = 8.3 Hz,

1H), 7.68 (d, J = 8.3 Hz, 1H), 7.40-7.28 (m, 7H), 7.13 (3H), 7.06 (d, J = 8.8 Hz, 1H), 6.92 (t, J = 7.3 Hz, 1H), 5.06 (AB kv, J = 12.2Hz, Dn = 8.9Hz, 2H), 4.18 (dd, J = 3.4, 8.8Hz, 1H), 4.10 (Quintet,

J = 6.8Hz, 1H), 3.57 (m, 2H), 3.50-3.22 (M, 3H), 2.62-2.37 (M, 2H), 2.23 (s, 3H). , 2.18-1.68 (m, 3H) 1.05 (d, J = 6.8 Hz, 3H) a minority peaks conformation) : δ 9.00 ( bs, 1H), 7.91 (bs, 1H), 7.84 (d, J = 8.3 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H) , 7.40-7.28 (m, 7H), 7.13 (3H), 7.06 1 (d, J = 8.8 Hz, 1H), 6.92 (T, J = 7.3Hz, 1H) 5.01 (ABkv, J = 12.2Hz, Dn = 19.0Hz, 2H), 4.32 (Dd, J = 2.4, 8.8Hz, 1H), 4.22 (quintet, J = ‘6.8Hz, 1H) 3.57 (m, 2H), 3.50-3.22 (m, 3H), 2.62-2.37 (m, 2H) , 2.23 (s, 3H), 2,18- -1.68 (m, 3H) 1.10 (D,

J = 6.8Hz, 3H);

¹³ C NMR (125 MHz _DMSO-d6) : mixture of rotamers, (spades prevailing conformation): δ 170.80 (0 = Ό), 170.52 (0 = 0), 169.18 (C = O), 152.61 (0 = 0), 138.10 (C) 137.38 (C), 136.04 (C), 130.05 (CH), 129.63 (CH), 129.47 (CH), 128.58 (C), 128.28 (CH), 127.89 (CH), 127.85 (C), 126.02 (CH), 122.50 (CH), 117.90 (CH), 117.81 (CH), 65.44 (CH ₂ ), 59.61 (CH), 47.04 (CH ₂ ), 41.75 (CH), 40,41 (CH ₂ ) 40.00 (CH ₂ ), 29.29 (CH ₂ ), 24.13 (CH ₂ ), 19.88 (CH ₃ ), 17.78 (CH ₃ ) and (peaks

minor conformation): δ 170.94 (0 = 0), 170.52 (C = O), 169.31

(C = O); 152.61 (C = O), 138.10 (C), 137.38 (C), 136.04 (C), 130.05 (CH), 129.63 (CH), 129.47 (CH), 128.65 (C), 128.28 (CH), 127.89 (CH), 127.85 (C), 126.02 (CH), 120.940 (CH), 117.90 (CH), 117.81 (CH), 65.44 (CH ₂ ), 59, 91 (CH), 46, 51 (CH ₂ ), 42.01 (CH), 40.13 (CH ₂ ), 39, 84 (CH ₂ ), 31.75 (CH ₂ ), . 22,11 (CH ₂ ), 20.05 (CH ₃ ), 17.78

(CH _3);

MS (EI) m / z 579 [M + Na] ⁺ 557, 454,426,357,336,293,267,201;

Elemental analysis for C 32 H 36 N 4 O 5: calculated C 69.05; H, 6.52;

N, 10.07; Found: C, 68.87; H, 6.52; N, 9.93.

Preparation of N - [[4 - [[(2-methylphenylamino) carbonyl] amino] phenyl] acetyl] -L-prolyl-3-methyl- (R) -β-alanine (8; oMePUPA-V)

A solution of 80.18 g of oMePUPA-V-OBn (7) in 800 ml of a 9: 1 mixture of tetrahydrofuran and water was hydrogenated at 50 psi hydrogen in the presence of 2.44 g of 10% palladium on carbon. After 25 hours, the reaction mixture is filtered through Solka Floc® (144 g; Fiber Sales & Development Corp.) on a fritted funnel. The filtrate was then filtered through another bed of Solka Floc® (115 g), evaporated to about 250 mL and poured into 3 L of well stirred toluene. The suspension is allowed to stir for 0.5 hours, filtered through a 2 L funnel with diatomaceous earth and the white powder obtained is first dried on a suction funnel (10.7 kPa; 0.5 hours) and then in a vacuum oven (14 hours; 45 hours). ° C; pressure adjusted to 3.33 kPa with nitrogen flow). The 3-piece lumps were crushed (mortar and pestle) to a fine powder to give 58.3 g (87% yield) of oMePUPA-V as a white solid. The product was recrystallized from acetone / water (320 mL / 75 mL). The crystals are filtered off and dried first in a suction fritted funnel (1 hour, 107 kPa) and then in a vacuum oven (25 hours; 45 ° C, pressure adjusted to 3.33 kPa with nitrogen flow) to give 47.0 g (84% after crystallization) oMePUPA-V as a white solid (HPLC purity 99.1%), mp 153.6-154.4 ° C;

IR (KBr) 3354, 3307, 1719, 1643, 1590, 1543, 1514, 1449, 1414,

1308, 1237 cm ^-1 ;

^H NMR (600 MHz, _DMSO-d6): mixture of rotamers 3: 2 (peaks for the predominant conformation): δ 12.21 (br s, 1H), 8.99 (s, 1H), 7.91 (s, 1H), 7.87 (d, J = 8.2Hz, 1H), 7.68 (d, J = 7.9Hz, 1H), 7.40 (d, J = 8.6Hz,

2H), 7.17 (d, J = 5.9 Hz, 2H), 7.15 (d, J = 7.6 Hz, 1H), 7.12 (dd,

J = 7.9, 8.2Hz, 1H), 6.94 (dd, J = 7.3, 7.3Hz, 1H), 4.22 (dd, J = 3.3,

8.8Hz, 1H), 4.06 (m, J = 6.6Hz, 1H), 3.47 (dd, 1H), 3.44 (d, J = 15.0Hz, 1H), 3.37 ( dd, 1H), 3.29 (d, J = 15.4Hz, 1H), 2.46 (dd, 1H), 2.27 (m, 1H), 2.25 (s, 3H), 1.99 (m, 1H); 1.80 (m, 1H);

1.78 (m, 1H), 1.76 (m, 1H), 1.07 (d, J = 6.6Hz, 3H) and (minor conformation peaks): Ô 12.21 (bs, 1H), 8.99 (s, 1H), 7.90 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 8.2 Hz, 1H), 7, 40 (d,

J = 8.6Hz, 2H), 7.16 (d, J = 5.9Hz, 2H), 7.15 (d, J = 7.6Hz, 1H), 7.12

(dd, J = 7.9, 8.2Hz, 1H), 6.94 (dd, J = 7.3, 7.3Hz, 1H), 4.34 (dd,

J = 1.8, 8.4Hz, 1H), 4.18 (m, J = 6.6Hz, 1H), 3.60 (m, 2H), 3.59 (m, 1H), 3.48 ( m, 1H), 2.47 (dd, J = 6.6, 15.4Hz, 1H), 2.40 (dd, J = 6.6, 15.4Hz, 1H), 2.25 (s, 3H) 2.15 (m, 1H); 1.83 (m, 1H);

1.91 (m, 1H); 1.77 (m, 1H); 1.12 (d, J = 6.6 Hz, 3H);

δ prevailing conformation):

59.7

137.5 ¹³ C NMR (150 MHz, DMSO-d ₆ ) (peaks for

172.4

(C = O), 170.9 (C = O), 169.3 (C = O), 152.6 (C = O); 138.2 (C), (C), 130.2 (CH), 129.8 (CH), 129 6 (CH) 128.7 (C), 127.4 126.1 (CH), 122.6 (CH), 120.9 (CH) , 118.0 (CH), 117.9 (CH), (CH), 46.6 (CH ₂ ), 41.7 (CH ₂ ), 40, 6 (CH ₂ ), 40.2 (CH ₂ ), 29.4

peaks for minor (CH ₂ ) conformation: δ 172.5 (C = O), 171.0 (C = O), 160.5 (C = O), 152.7 (C = O), 138.19 ( C), 137.5 (C), 130.2 (CH), 129.8 (CH), 129.6 (CH), 128.8 (C), 127.4 (C), 126.1 (CH) ), 122.6 (CH), 120.9 (CH), 118.0 (CH), 117.9 (CH), 59.9 (CH), 47.1 (CH ₂ ), 42.0 (CH) ₂ ), 39.8 (CH ₂ ), 40.3 (CH ₂ ), 31.8 (CH ₂ ), 24.2 (CH ₂ ), 20.2 (CH ₃ ), 17.9 (CH ₃ ) ;

MS (EI) m / z 468 [M + H] ⁺ , 336, 267, 137;

H, C ₂ 5H3oN Person _4: C, 64.36; H, 6.48; N, 12.01; Found: C, 64.07; H, 6.40; N, 11.85.

Example 2

Activity in a sheep model of allergic pneumonia

Allergic sheep weighing 27 to 50 kg shall be used.

The development of both early and late bronchial responses to the sprayed allergen Ascaris suum was first demonstrated in all sheep. The sheep were conscious and kept in a modified shopping trolley lying flat with their immobilized head. After local anesthesia of the nasal cavity with 2% lidocaine, a balloon catheter is inserted through the nostrils into the lower part of the esophagus. Animals are intubated with a fixed endotracheal tube through a second nostril using a flexible fiber optic bronchoscope as a guide. All procedures used in this study are agreed by the Mount Sinai Medical Center of the Animal Research Committee, which is responsible for ensuring the humane handling and use of experimental animals.

·······················································

Pleural pressure is adjusted using an esophageal balloon catheter (filled with 1 ml of air) which is placed 5-10 cm from the gastroesophageal junction. In this position, the terminal expiratory pleural pressure is in the range of -2 to -5 cm H ₂ O. Once the balloon is placed, it is ensured that it remains in the same position throughout the experiment. Lateral pressure in the respiratory tube is measured by a catheter with a lateral orifice (2.5 mm ID) inserted through the endotracheal tube and placed distally at the end of the endotracheal tube.

Tracheal and pleural pressure catheters are connected to a differential pressure transducer (MP45, Validyne, Northridge, CA) to measure transpulmonary pressure, which is defined as the difference between tracheal and pleural pressure. Air flow is measured by attaching the proximal end of the endotracheal tube to a pneumotachograph (Fleisch, Dynea Science, Inc., Blue Beli, PA). Transpulmonary pressure and flow signals are recorded on a multichannel physiological recording device, which is connected to a personal computer 80-386 DOS for on-line calculation of mean lung flow resistance (R _L ) by dividing the change in transpulmonary pressure by changing the mean inspiratory volume flow (obtained by digital integration) . To obtain R _L in cmH ₂ O / l / sec. a minimum of 5 breaths shall be used, with no swallowing artifact. Immediately after the measurement of R _L was measured thoracic gas volume (V _tg) within a body plethysmograph with a constant volume to give a specific lung resistance (SR _L = R _L x V _tg) in L x _cmH2O / L / sec. .

All liquid dosed aerosols are generated using a medical air nebulizer (Raindrop ®, Puritan Bennett, Lenexa, KS), which provides an aerosol with a mean aerodynamic diameter of 3.2 µm, as determined by an Andersen cascade impactor. The nebulizer is connected to a dosimetric system consisting of a solenoid valve and a source of compressed air (137.9 kPa). The outlet of the nebulizer leads to a T-shaped plastic piece, one end of which is connected to the inhalation device. • ······························ • Piston Respirator Port (Harvard Apparatus, S. Natic, MA). The solenoid valve is activated for one second at the start of the respirator inhalation cycle. The aerosol is delivered in a respiratory volume of 500 ml and at 20 breaths per minute. To assess bronchial sensitivity, cumulative response curves to carbachol concentration are generated by measuring SR _L immediately after inhalation of the buffer and after each subsequent administration of 10 breaths of increasing carbachol concentration (0.25, 0.5, 1.0, 2.0, and

4.0% w / v in buffered saline). The irritation test is terminated when SR1 rises above 400% of the value after saline administration or after the highest concentration of carbachol. Bronchial sensitivity is assessed by determining the cumulative concentration of carbachol (in the breath unit), which increases SR _L by 400% relative to saline (PC ₄₀ ) by interpolation of the dose-response curve. One breath unit (BU) is defined as one breath of a 1% (w / v) spray solution of carbachol.

Doses of oMePUPA-V are dissolved in either a 1: 2 mixture of ethanol and normal saline, a 1: 5 mixture of ethanol and 200 mM sodium phosphate or Tris buffer. When Tris is used, any desired dilution is prepared using normal saline. Doses are prepared in a total volume of 3-5 ml.

In all studies, the baseline airway sensitivity line (i.e. PC ₄ oo) is determined three to four days before the start of the study. In a single dose preliminary study, SR _L is measured and animals are treated with the compound or vehicle. SR _L is measured 2 hours after treatment (just before stimulation) and then the animals are stimulated with an allergen. In a multi-dose study, starting 4 days prior to allergen challenge, animals are treated once daily for 4 days and stimulated with allergen 24 hours after the last dose. SR _L was measured, before and after the last dose of compound or vehicle. In all studies, SR _{L is} measured immediately after allergen stimulation, after 1 to 6 hours after stimulation and after half an hour.

• · ·· ···· · · ··

6.5 to 8 hours after allergen stimulation. Determination of airway sensitivity (PC400) after stimulation is performed 24 hours after allergen stimulation.

Values are expressed as mean ± standard deviation of the mean. Change of SR L was calculated for each sheep as the difference with the baseline SR _L before the test. SR _L post-challenge changes are characterized by an early airway response (EAR) that develops over a period of 0 to 4 hours. This is followed by a late airway response (LAR) that develops about 4 to 8 hours after allergen stimulation. The areas under the EAR and LAR curves are calculated for each animal using a trapezoidal rule. A significant reduction in area under the EAR and LAR curves compared to placebo control is considered a therapeutic effect on change in SR _L allergen-induced. The airway sensitivity to carbachol (PC400) tested before and 24 hours after allergen challenge is expressed as the ratio of PC400 (post-pretreatment PC400 value) to each sheep. A significant increase in the ratio of PC400 relative to placebo control is considered a therapeutic effect. Comparison with the placebo control was performed using a one-way analysis of variance, followed by Dunnett's test (one-sided) for multiple comparison with the control. Comparisons leading to p <0.05 are considered statistically significant.

Fig. 1 shows an aerosol-inhibiting dose response of oMePUPA-V in Ascaris suum sensitized sheep 2 hours after dosing. The left panel shows the change in SR lung specific resistance, ₁ cm HsO / sec. The right panels show airway sensitivity to inhaled carbachol (PC400 ratio, pre / post stimulation) found 24 hours post-stimulation. oMePUPA-V at doses of 0.01 to 0.03 mg did not show early or late respiratory responses or did not alter carbachol hypersensitivity 24 hours after allergen stimulation. Doses of 0.1, 1 and 3 mg inhibit early airway response and maximally inhibit late airway response. These doses also inhibit carbachol hypersensitivity 24 hours after stimulation. ········· Statistical analysis of these data is presented in Table 2.

Table 2

treatment Dose (mg) vehicle n EAR (ASR _L xh) LAR (ASRLx) PC400 (after / before) Dose 2 hours before allergen challenge PBS 12 5.85 + 0.62 4.85 ± 0.69 0.49 ± 0.03 OMePUPA-V 0.01 EtOH: NS 2 6.87 ± 0.05 5.11 ± 1.46 0.44 ± 0.04 0.03 EtOH: NS 2 10.62 ± 3.91 3.98 ± 0.23 0.43 ± 0.00 0.10 EtOH: NS 4 2.54 ± 0.74 * 0.67 ± 0.17 * 0.18 ± 0.11 * 1.00 EtOH: PBS 2 2.14 + 0.70 0.27 ± 0.34 * 1.05 ± 0.11 * 3.00 EtOH: PBS 2 2.47 + 0.62 0.68 ± 0.07 * L, 07 ± 0.08 *

= p <0.05 vs. PBS control, single-factor analysis of variance, followed by Dunnett's test for multiple comparison with the control group. It shows a statistically significant decrease in EAR or LAR or a significant increase in the ratio of PC400 relative to the PBS control group.

Sheep naturally sensitive to Ascaris suum are stimulated with an Ascaris suum allergen aerosol 2 hours after oMePUPA-V aerosol administration at the indicated doses or 24 hours after the last dose of oMePUPA-V repeated dosing for four days lower than baseline or equivalent amount of PBS. Lung activity, referred to as change in specific airway resistance relative to baseline value prior to study, is measured 8 hours after allergen challenge. Early airway response (0-4 hours, EAR) and late airway response (4-8 hours, LAR) are expressed as mean area under the lung specific resistance curve versus time ± standard deviation of mean. The airway resistance to inhaled carbachol is determined before the start of the study and 24 hours after allergen stimulation. Respiratory sensitivity is reported as the ratio of PC400 (the amount of carbachol needed to increase resistance by 400%) by comparing values before and after stimulation.

• ····························································· ·· ·· ···· ·· ·

Single dose irritation

None of the doses of oMePUPA-V used in the above study have an irritant effect, as is evident from the fact that there is no change in airway resistance relative to the baseline resistance after Ascaris suum allergen stimulation. The results are shown in FIG. Second

Repeated dose studies

Fig. 3 illustrates that a 0.03 mg dose of oMePUPA-V, which has been shown to be ineffective when used in single dose pretreatment, is effective when administered once daily for 4 days when antigen stimulation occurs 24 hours after the last dose . The upper and lower left panels show that this effect was evident using two different formulations. Hypersensitivity to carbachol after an additional 24 hours was also maximally inhibited, as seen from the upper and lower right panels in Figure 3. The protective effect of oMePUPA-V was significant against EAR and LAR and against carbachol hypersensitivity and quantitative analysis is shown in Table 3.

The results of this study show that single pretreatment with a small molecule VLA inhibitor, oMePUPA-V, in the form of an aerosol can protect against allergen-induced early and late respiratory responses after allergen irritation in an allergic sheep model. There is no respiratory irritant effect at any doses of oMePUPA-V given individually during pretreatment. The results also showed that the effective dose of oMePUPA-V can be reduced with multiple treatments. Together, these data provide clear evidence that the VLA-4 adhesion pathway plays a critical role in the pathophysiological indicators (LAR and AHR) of the longer inflammatory conditions that are induced in the airways of allergic sheep after allergen irritation.

• ······································ · ··· ··· ·· ···· ·· ···

Table 3

treatment Dose (mg) vehicle n EAR (ASR _L xh) LAR (ASR _L xh) PC4Q0 (after / before) Dose once daily for 4 days, allergen stimulation 24 hours after the last dose PBS 8 4.33 ± 0.81 4.96 ± 0.40 0.38 ± 0.03 oMePUPA-V 0.03 EtOH: NS 4 L, 53 ± 0.34 * 0.59 ± 0.16 * L, 06 ± 0.04 * 0.03 Tris: NS 4 1.40 + 0.35 * 0.02 + 0.06 * 1.04 ± 0.04 *

* = p <0.05 vs. PBS control, single-factor analysis of variance, followed by Dunnett's test for multiple comparison with the control group. It shows a statistically significant decrease in EAR or LAR or a significant increase in the ratio of PC400 relative to the PBS control group.

Sheep, naturally sensitive to Ascaris suum, are stimulated with an Ascaris suum allergen aerosol 24 hours after the last dose by repeated daily administration of a lower dose of oMePUPA-V than baseline, or an equivalent amount of PBS for 4 days. Pulmonary mechanics, referred to as change in specific airway resistance relative to baseline before study, is measured 8 hours after allergen challenge. The early airway response (0-4 hours, EAR) and the late airway response (4-8 hours, LAR) are expressed as the mean area under the specific lung resistance curve versus time ± standard deviation of mean. Airway resistance to inhaled carbachol is determined before the start of the study and 24 hours after allergen stimulation. Respiratory sensitivity is reported as the ratio of PC400 (the amount of carbachol needed to increase resistance by 400%) by comparing values before and after stimulation.

Example 3

Activity on delayed type hypersensitivity model

Studies in a model of sheep red blood cells

Female mice of a specific species are used for all experiments. · · · · ··· ·· ···· ·· ···

Patbogen-free Balb / c 8 to 10 weeks of age from Jackson Labs. Animals are fed and watered to taste. Sheep red blood cell (sRBC) cells in Alsever solution from the same sheep are obtained weekly from Charles River Pharm. Services (Southbridge, MA). The sRBCs are pelleted by centrifugation at 1000 g for 10 minutes at 4 ° C and any visible coating is removed. The cells are then washed with saline. The cell pellet is suspended in saline and counted using a hemocytometer. Cells are diluted in phosphate buffered saline (PBS) to 2x10 ⁸ sRBC per ml. On day 0, mice are sensitized with

sc injection of 2x10 ⁷ sRBC in 100 μΐ PBS. On day 5, sRBC is prepared as above, but diluted in PBS to a final concentration of 4x10 ⁹ sRBC per ml. From this preparation, 25 μΐ is injected sc into the foot of the right hind limb.

For enteral administration of the compound, oMePUPA-V (Lot # 2770-029) is formulated in a vehicle containing 60% PEG 400 in 0.02 M TRIS to give a stock solution at a concentration of 5 mg / ml. Appropriate dilutions are prepared in a vehicle containing PEG / TRIS and administered enterally in a volume of 100 μΐ. Anti-VLA-4 antibody (PS / 2) is diluted in saline at a concentration of 4.3 mg / kg and administered intraperitoneally in a volume of 100 μΐ. All drugs are administered immediately after sRBC stimulation.

Swelling of unstimulated (left) control limb and stimulated (right) hind limb was measured using a Mitutoyo CD caliper (Model # 304-196, Dyer, Lancaster, PA) 20 hours after limb stimulation. Data are expressed as a change in the thickness of the foot, which is determined by subtracting the thickness of the left hind limb and the right hind limb. Changes in foot thickness are compared using a double-sided Student's t-test.

The anti-VLA-4 antibody PS / 2 at 4.3 mg / kg intraperitoneally inhibits swelling by about 30%, while oMePUPA-V administered enterally at 20 mg / kg has no effect in this model (data). · ···································································· ·· ·· ··· not listed). The efficacy of oMePUPA-V administered at 20 mg / kg by the enteral route in the sRBC-induced DTH model in mice was studied and no effect was found.

Example 4

Activity on delayed type hypersensitivity model

Twenty-gram female Balb / c virus-free mice (Jackson Laboratories, Bar Harbor, ME), placed four per cage in micro-insulated cages in a Biogen-free device, are used for all studies and are fed a mouse feed with a water tap. . Mice are anesthetized with ketamine: xylazine (90:10 mg / kg, ip). 3 cm ² of abdominal skin is shaved and the skin is washed with 70% ethanol. 25 μΐ 0.5% DNFB in a 4: 1 v / v mixture of acetone and olive oil as a vehicle is uniformly applied to the bare spot. The skin is easily scratched by the tip of the pipette, causing a slight inflammation. The mouse is placed back in its cage and allowed to recover from anesthesia. 24 hours later after the initial sensitization, the mice are sensitized again with 25 μΐ 0.5% DNFB in the same vehicle on the abdomen skin, followed by a slight scratch with the pipette tip. In the second sensitization, the mice are not anesthetized. On day five (about 120 hours after the first sensitization), a stimulating dose of sensitizer (0.2% DNFB in a 4: 1 (v / v) acetone / olive oil mixture as vehicle) is used to stimulate the immune response. Mice are anesthetized with 90:10 mg / kg ketamine: xylazine ip and 10 μΐ 0.2% DNFB is applied to the back of the left ear. A 4: 1 v / v mixture of acetone and olive oil is applied to the right ear as a vehicle. In the next 24 hours develops a biphasic ear swelling response, as shown in Figure 4. 24 hours after challenge mice were again anesthetized with ketamine: xylazine and measured them with a caliper thickness of both ears to within 10 ^"4 inches (0 , 000254 cm).

Orally, the probe is administered 4 hours after immune stimulation. Day 5 responses are administered by compounds (100 μΐ) or a suitable vehicle (dimethylsulfoxide [DMSO] in isotonic phosphate buffered saline [PBS], 100 μΐ). A group of 8 mice is used for each test. oMePUPA-V (batch number 2044-076) is dissolved in distilled water by the addition of 0.5% sodium phosphate buffer, pH 8.8 and 3% dimethylsulfoxide. Ear swelling for each mouse is calculated as the difference between the thickness of vehicle-stimulated ear and DNFB 24 hours after stimulation. A typical DNFB-induced ear swelling is 65-75x10 ^-4 inches. Ear swelling inhibition is determined by comparing the treatment group with the vehicle treated group. The statistical significance of the difference between treatment groups is evaluated using a single-factor analysis of variance, followed by Dunnett's test for multiple comparison with the control group (Systat, SPSS Inc.) using p <0.05. Values are expressed as mean ± SEM.

Fig. 4 compares the ear swelling measured 24 hours after DNFB stimulation in mice receiving vehicle (DMSO, PBS), a positive control compound (administered at 0.03 µg / kg) or 0.03 or 0.3 mg / kg oMePUPA-V, administered enterally 4 hours after DMFB stimulation (upper panel). The percent inhibition caused by treatment is shown in the lower panel. Both doses of oMePUPA-V significantly inhibit ear swelling to a similar extent to that of the positive control compound.

A single enteral dose of 0.03 or 0.3 mg / kg oMePUPA-V administered 4 hours after DNFB stimulation can significantly inhibit ear swelling in a mouse contact hypersensitivity model.

Example 5

biochemistry

1.5 OMePUPA-V receptor affinity as measured by VCAM-Ig alkaline phosphatase conjugate in VCAM-Ig direct binding assay (DBA)

The VCAM-Ig is prepared and purified according to the procedure described in the following: &lt; RTI ID = 0.0 &gt; 1 &lt; / RTI &gt; • (Jakubowski, A., et al. Cell Adhesion and Communication 3: 131-142; 1995). Conjugation to calf intestinal phosphatase (to cleave the chromogenic substrate) is performed according to the procedure of Lobb RR et al., Cell Adhesion and Communication 3: 385-397, 1995). VLA binding is tested on a human T cell line, Jurkat (α4β1). VCAM-Ig-AP and oMePUPA-V compete for binding to α4β1 on the surface of these cells in the presence of 1 mM Mn ²⁺ .

In the VCAM-Ig direct binding assay, oMePUPA-V competes with VCAM-Ig-AP for binding to Jurkat cells in the presence of 1 mM manganese chloride concentration versus IC50 of 8 ± 1 nM (n = 9). The results are shown in Table 4.

5.2. Affinity of the oMePUPA-V receptor as measured by VCAM-Ig alkaline phosphatase conjugate in purified VLA-4 protein / protein assay

VLA-4 is purified from a detergent extract of a highly expressing subclone of α4 transfected K562 cells by antibody affinity chromatography and immobilized to microtiter wells to prepare a protein / protein competitive binding assay. VCAM-Ig-AP binds to a plate coated with purified VLA-4 in the absence or presence of oMePUPA-V (Lot # 2) and 1 mM MnCl 2 - Plates are read at 405 nm and data are analyzed using SoftMax v software. 2.32.

Binding of VCAM-Ig conjugate to purified VLA-4 is totally blocked by specific neutralization of anti-α4 monoclonal antibody (HP1 / 2). The two IC50s obtained for oMePUPA-V in the VLA-4 protein / protein assay are shown in Table 4, as are IC50 values obtained on Jurkat cells from the VCAM-Ig -AP competitive binding assay and the CS1 cell adhesion assay.

3.5 OMePUPA-V receptor affinity tested in CS-1 cell adhesion assay

a. Adhesion of Jurkat Cells to CS1 / BSA Conjugate 9 · 9 · 9 · 9

The NH2-cysteine-tyrosine-CS-1 peptide is synthesized and condensed to BSA-SMCC (SMCC is a heterobifunctional crosslinker that reacts with free amino groups on BSA and cysteine synthetic peptide alone) to CS1 / BSA in a 10: 1 ratio. The wells are coated overnight with 100 μΐ of conjugate diluted to a final concentration of 1 μg / ml. The next day the wells are blocked with BSA in PBS for one hour and then washed three times.

The human T-cell strain, Jurkat cells, is designated 2 μΜ BCECF-AM (fluorescent dye (2 ', 7'-bis (2-carboxyethyl) -5) and

6) carboxyfluorescein acetoxymethyl ester (Molecular Probes Inc., Eugene, Oregon; Cat. # B-1150)), which is incorporated and de-esterifies the dye-binding sites by living cells. Jurkat cells (1x10 ⁵ cells / well) in buffer containing 1 mM Mn ²⁺ are added to the coated plates in the presence of triplicate serial dilutions of inhibitor. Each concentration is tested in duplicate. After 30 minutes at room temperature, the plates are inverted and washed three times, or until no cells are adhered to control wells coated with BSA alone. CS1-adherent cells are quantitated in a Cytofluor fluorescence plate reader using an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Cells adhering to CS1 / BSA in the absence of compound serve as a 0% inhibition control, while cells adhering to BSA alone serve as a 100% inhibition control. IC 50's are calculated using Deltagraph software, version 5.

Adhesion of labeled Jurkat cells in the presence of Mn ²⁺ is completely blocked by EDTA and neutralized by the anti-α4β1 mAb, HP1 / 2, confirming that binding is specific. Table 4 contains the oMePUPA-V activity in the CS1 / BSA adhesion assay as well as the binding assay results.

oMePUPA-V is a potent VLA-4 antagonist in a buffer containing

Mn. The severity test is eighty times more effective when tested in the presence of Mn ²⁺ on isolated VLA-4 than on Jurkato • B

BB B B 8 B B B BB

B · B · · BB

BBB BBB BB BBBB BB B cell. oMePUPA-V is a functional antagonist, as evidenced by its ability to block dose-dependent and total adhesion of Jurkat cells to CS1. The absolute values in the adhesion test are higher than those observed in the severity test. This may be due to the multivalent nature of adhesion. In any assay format, inhibition of binding to EDTA and HP1 / 2 demonstrates specific binding to VLA-4.

Table 4

OMePUPA-V receptor affinity in the presence of 1 mM MnCl ₂ according to the VCAM-Ig competition binding assay, CS1 cell adhesion assay and purified VLA-4 protein / protein assay

IC ₅₀ ± SD Test oMePUPA-V Binding of Jurkat cell VCAM-IG 8 + 1 (n = 9) Adhesion of Jurkat cell CS1 22 + 2 (n = 4) Binding of purified VLA-4 VCAM-Ig 0.1 (n = 1) (0.1, 0.1)

4.6 Specificity of oMePUPA-V inhibition

a. The specificity of oMePUPA-V is tested by JY cells in the VCAM-Ig direct binding assay and the CS1 adhesion assay.

Α4β7 binding is tested on JY cells in the presence of Mn ²⁺ . In the VCAM-Ig and oMePUPA-V binding assay, they compete for binding to α4β7 on JY cells (see section 4.1.1 for assay procedure). The cell adhesion assay tests the ability of oMePUPA-V to block JY (α4β7) binding of cells to the CS1 / BSA conjugate.

oMePPA-V does not block α4β7 binding to VCAM-Ig or CS1 / BSA. Αηίί-β7 Mab, Fib27 (Pharmingen), completely inhibits these interactions, suggesting that the binding was α4β7 specific. Therefore, oMePUPA-V is a specific inhibitor of VLA-4. The results are shown in Table 5.

b. Specificity of oMePUPA-V, tested by adhesion of K562 cells to Fn-120 coated wells

Untreated 96 well flat bottom polystyrene plates were coated overnight at 4 ° C with 5 gg / ml Fn-120. Plates are washed twice with phosphate buffered saline (PBS) and blocked for 1 hour at room temperature with 1% bovine serum albumin (BSA). Plates are washed twice with TBS buffer containing 1 mM MnCl ²⁺ (assay buffer). Label K562 cells with 2 μΜ of fluorescent dye, BCECF-AM (see section 4.1.3) and bind to the plate at room temperature for 30 minutes. Plates are inverted and washed three times, and adherent cells are quantified in a fluorescent plate reader Cytofluor using an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

The adhesion of K562 to Fn-120 is completely blocked by neutralizing the anti-α5 antibody, IIA1 (Pharmingen), indicating specific binding through VLA-5. Inhibition of K562 cells binding to the Fn 120K fragment of oMePUPA-V does not occur at doses of 100 μΜ. See Table 5 below.

c. Aggregation assay to determine the specificity of oMePUPA-V

ways

Activity against gpIIbIIa is assayed by standard platelet aggregation using platelet-rich plasma. ADP is used to induce aggregation in the presence of plasma, where Ca ²⁺ and Mg ^{2+ are the} major bivalent cations. GRGDSP @ 100 μg / ml is used as a positive control.

Results oMePUPA-V is tested at three doses of 1, 10 and 100 μΜ. It does not inhibit ADP-induced platelet aggregation at any dose. The results are shown in Table 5. oMePUPA-V is very (> 10,000 times) specific for VLA-4. There is no measurable activity (> 100 μΜ) against the related integrins α4β7 and VLA-5. • or against integrin β3, gpllbllla.

Table 5

OMePUPA-V inhibitory activity according to α4β7 VCAM-Ig competitive binding assay, α4β7 and VLA-5 adhesion assays and platelet aggregation studies

Cell strain ligand Divalent cation oMePUPA-V [NM] ic ₅₀ ± sd JY α4β7 VCAM-Ig DBA Mn ²⁺ 3% inhibition @ 100 μΜ (n = 3) JY α4β7 CS1 / BSA adhesion Mn ²⁺ no @ 100 μΜ (N = 4) inhibition K562 (VLA-5) Fn-120 adhesion Mn ^{of +} no @ 100 μΜ (N = 3) inhibition inserts (IlblIIa) fibrinogen aggregation Ca ²⁺ / Mn ²⁺ no @ 100 μΜ (N =) inhibition

Example 6

OMePUPA-V assay for LIBS induction

6.1. Jurkat cell measurements using LIBS antibody 9EG7

a. The induction of LIBS by α4β1 antagonists is tested in vitro by FACS analysis. Jurkat cells (2x10 ⁵ / well) are preincubated at 37 ° C for 20 minutes with TRIS buffered saline containing 2 mM MgCl 2 (Mg ²⁺ -TBS) alone or from a series of dilutions of the test compound. The samples are transferred to an ice bath and supplemented with LIBS antibody, 9EG7, resulting in

O concentration of 10 μg / ml. Cells are washed twice with Mg-TBS and suspended in a 1: 200 dilution of FITC-conjugated goat anti-rat IgG in Mg ²⁺ -TBS and incubated for 30 minutes at 4 ° C. The cells are washed twice and resuspended in Mg ²⁺ -TBS. The average fluorescence intensity (MFI) is determined by FACS analysis (Becton Dickinson FACScan). The results are expressed as MFI. Data is analyzed using Microsoft Excel version 5.0

• and · Deltagraph version 4.0.

Fig. 5 shows that oMePUPA-V induced LIBS epitope exposure compared to 2 mM Mg ²⁺ buffer (panel B). The induction was concentration-dependent and had similar magnitude to that observed with 1 mM Mn ²⁺ (panel A). Omission of LIBS antibody and antibody detection, or omission of antibody detection alone eliminates labeling (panel B). The ED50 response values are 20 nM.

conclusion

These data show that oMePUPA-V induces the same conformational change in VLA-4 as seen with native ligands. LIBS values generally fall within the defined binding and adhesion assays, which are 8 nM and 22 nM, respectively, for oMePUPA-V.

2.6 Panel of receptors from different species

a. OMePUPA-V receptor affinity according to VCAM-Ig direct binding assay using VCAM-Ig alkaline phosphatase conjugate and peripheral blood lymphocytes or spleen cells from different species

PBLs are isolated from human, sheep and dog peripheral blood using the methods described for sheep PBL (Abraham W.M. et al., J. Clin. Invest., 93: 776-787, 1991). A competitive VCAM-Ig-AP binding assay is used to compare the binding of oMePUPA-V to these different cell types.

The IC 50 values obtained for oMePUPA-V on peripheral blood lymphocytes or spleen cells from different species in the presence of Mn ²⁺ are shown in Table 6. In the presence of Mn ²⁺ , oMePUPA-V inhibits the binding of VCAM-Ig to human-derived lymphocytes rats, dogs, sheep and mice. There is therefore no evidence of species specificity. This result is in line with the high degree of sequence conservation observed between the species for VLA-4 and its natural ligands, CS-1 and VCAM.

··············································

Table 6

OMePUPA-V receptor affinity according to VCAM-Ig competition binding assay using VCAM-Ig alkaline phosphatase conjugate and peripheral blood lymphocytes or spleen cells from different species

Types source Divalent cation IC ₅₀ [nM] Man PBL Mn ²⁺ 6 + 1 (n = 3) sheep PBL Mn ²⁺ 3 + 1 (n = 3) pig PBL Mn ²⁺ 13 + 2 (n = 3) mouse splenocytes Mn ²⁺ 4 + 2 (n = 4) rat splenocytes Mn ²⁺ 5 + 1 (n = 3)

Example 7

OMePUPA-V receptor kinetics

7.1. Competition assay using ³ H-known inhibitor as probe

Jurkat cells are maintained in RPMI-1640 medium plus 10% fetal bovine serum at 37 ° C in a tissue culture incubator. For binding studies, cells are pelleted by centrifugation, washed twice with TBS (50 mM Tris HCl, 150 mM NaCl, 0.1% bovine serum albumin, 2 mM glucose, 10 mM HEPES pH 7.4), suspended at about 2x10 ⁶ cells / ml in TBS and counted using a Neubauer hemocytometer. Cells are further diluted in labeled buffers to 1.5x10 ⁶ / ml and subjected to the treatment described for each experiment. The cells are then pelleted by centrifugation, resuspended in

100 μΐ of assay buffer and transfer containing 2,9 ml

ScintiVerse II is scintillated (Fisher Scientific).

container

Radioactivity related to cells with fasting counters.

The number of bonds quantitates under these scintiintegrin, measured by the compound using conditions, and is not occupied, therefore, free to bind to the ³ H known inhibitor. All studies tested are performed in a siliconized 1.5 ml eppendorf tube at a standard sample volume of 1 ml. Non-specific binding of the ³ H-known inhibitor to the cells (background) is defined by measuring the binding of the inhibitor in the absence of a metal ion. Number of Specific Links ··· ········································ calculated by subtracting non-specific bonds from the total number of bonds.

A series of competitive studies is performed to verify that oMePUPA-V and a known inhibitor compete for the same site on VLA-4. First, the ³ H-known inhibitor is mixed with an equimolar amount of oMePUPA-V, a 10-fold excess and a 100-fold excess, incubated with Jurkat cells and the ability of the cold (unlabeled) inhibitor to compete for binding of the known inhibitor. Treatment with oMePUPA-V provides dose dependent inhibition of ³ H-known inhibitor binding. The concentration of oMePUPA-V required to compete for binding of the ³ H-known inhibitor is ten-fold higher than that required when a cold inhibitor was used as a competitor, corresponding to its lower affinity for Mn ²⁺ activated VLA-4. Second, Mn ²⁺ -activated Jurkat cells are treated with a ³ H-known inhibitor to first occupy a VLA-4 radioactive probe, and then an excess of cold oMePUPA-V is added. Upon treatment with an excess of cold oMePUPA-V or a known inhibitor, it was not possible to distinguish the ability to replace the radioactive probe. Third, Mn ²⁺ -activated Jurkat cells are treated with a saturating amount of oMePUPA-V and the rate at which oMePUPA-V dissociates are measured. In contrast to the extended half-life of the known inhibitor of Mn ²⁺ -activated VLA-4, oMePUPA-V is rapidly released from oMePUPA-V-VLA-4 at t 1/2 of less than 10 minutes. The large difference in t _1/2 for oMePUPA-V and the known inhibitor suggests that the lower affinity of oMePUPA-V for VLA-4 is due to its higher release rate.

The dissociation data show that the binding of oMePUPA-V to VLA-4 is highly dependent on the activation state of VLA-4 and that it exhibits the same selectivity for activation as the known inhibitor. As with Mn ²⁺ -activated VLA-4, t 1/2 dissociation of oMePUPA-V from Mn ²⁺ -activated VLA-4 was less than 10 minutes, which is the shortest period of time that can be evaluated in a competitive format. On the other hand, in the presence of Mg ²⁺ plus activating antibody, TS2 / 16, was t 1/2 longer (20 minutes). All possible activation states ······················································································ · ·· · have not been tested in detail, but a simple overview test has been carried out that can quickly clarify the differences. In this assay, a given concentration of oMePUPA-V (10 nM) is mixed with 5 nM ³ H-known inhibitor, and binding under these conditions is performed at various activation states. When oMePUPA-V has an extremely high or low affinity for VLA-4, this condition can be ascertained by a difference in the amount of ³ H-known inhibitor. The percent differences in the binding of the ³ H-known inhibitor under different activation conditions suggest, which can be estimated based on the known properties of the inhibitor.

Binding studies confirm that oMePUPA-V competes with a known inhibitor of binding to VLA-4 at concentrations that correspond to its affinity and demonstrates that the two compounds compete for the same site on integrin. The similarity of binding of oMePUPA-V to a known inhibitor at different VLA-4 activation states suggests that the binding mechanism is similar.

2.7 OMePUPA-V Assay on Panlabs and Cerep Panels oMePUPA-V is tested on radioligand, enzyme, and functional assays using the Panlabs ProfilingScreen, DiscoveryScreen and Immunoscreen Assay Kit and the Cerep Membrane Receptor Panel Assay Kit. For oMePUPA-V, no significant activity was observed in any assay involving the NK1 receptor assay at 10 μΜ, while known inhibitors show some activity.

Cerep also shows that oMePUPA-V does not inhibit human ACE protease activity. The source of ACE proteases was human endothelial cells (HUVEC). ³ H-HGG, added to HUVEC, is converted to ³ H-hipuric acid and glycylglycine by ACE. Captopril, a potent ACE inhibitor, blocks conversion at an IC 50 of 990 pM, while oMePUPA-V does not block it at 10 µΜ.

Pharmaceutical properties:

oMePUPA-V is an off-white crystalline powder. It is soluble in dimethylsulfoxide and has a water solubility of 0.120 mg / ml. Behavior ····························· In the hot, DSC, TGA and hot microscopy were studied and the substance was found to melt at about 160 ° C. DSC and TGA analysis revealed that at about 136 ° C oMePUPA-V loses volatile impurities and is likely to dehydrate the monohydrate.

formulations

Nebulization formulation

Further, a production resulting in a 100 ml nebulization formulation containing 5 mg / ml oMePUPA-V is described.

Prepare 200 ml stock buffer solution:

1. Weigh 0.286 g of Tromethamine, USP into a suitable container.

2. Weigh 1.676 g of sodium chloride, USP, into a suitable container.

3. Add 200 mL of water for injection, USP.

The mixture is mixed to make it homogeneous.

1. Weigh 0.500 g of oMePUPA-V into a suitable container.

2. Add 100 ml of the buffer solution prepared in step 1.

3. Mix until homogeneous.

4. Sterile filtration into a suitable container.

5. Seal with a suitable closure.

Typical formulation characteristics:

pH: 7.4, osmolality: 290 mOsm

Example 9

Stability Test Procedure by HPLC

Column: Zorbax® SB-C18, particle size 3 µm, 4.6x150 mm

Protective column: Zorbax® SB-C18, particle size 5 µιη, 4.6x12.5 mm Flow rate: 1 ml / min.

Column temperature: 40 ° C

Autosampler temperature: 4 ° C

Mobile phase:

A: 0.1% (w / v) trifluoroacetic acid (TFA) in water ··················· ·· ···· ·· ·

B: 0.1% (w / v) trifluoroacetic acid (TFA) in 90% (v / v) acetonitrile, 10% (v / v) water

gradient:

Time (min)% B

Delta-315

3-18 15 to 100

18-21100

21-2815

Injection: 10 μΐ 0.2 mg / ml solution in Tris / NaCl / water (intermediate) or 0.1 TFA / 45% acetonitrile (final product)

Detection: UV 254 nm (primary) and 215 nm

Control: oMePUPA-V, heat in boiling water for 20 minutes.

Pre-qualification was performed.

Stability of the drug substance:

Storage of the intermediate in bulk for two weeks under the following conditions showed no degradation: at 40 ° C in a sealed vial; at 50 ° C in a sealed vial; at 25 ° C, 60% RH; and at 40 ° C, 75% RH. At week four, one or two detectable degradation peaks occurred at 40 ° C and 50 ° C, but the level of impurities was still lower than 0.02%.

Stability of solution

a) A nebulizing formulation, 5 mg / ml in Tris / NaCl, stored at room temperature for two months, showed early eluted degradation peaks at a total of 1% (at 254 nm) and 2-3% (at 215 nm).

b) Heating the nebulizing form in boiling water for 20 minutes reduced the purity from 99.9% to 98.7% at 254 nm and from 100% to 93.6% at 215 nm.

c) A solution of 0.2 mg / ml in Tris / NaCl / water at neutral pH is stable at 2-8 ° C for at least one week.

• ··································· ··· ·· ···· ·· ·

It will be apparent to those skilled in the art that various modifications and changes may be made to the claimed invention without departing from the spirit and scope of the invention. It is intended that the invention encompasses modifications and variations of the invention provided they fall within the scope of the appended claims and their equivalents.

Claims (13)

PATENT CLAIMS A compound which inhibits cellular adhesion of oMePUPA-V and pharmaceutically acceptable esters, salts or isomers thereof. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 2, further comprising a second agent selected from the group consisting of corticosteroids, bronchodilators, antiasthmatics, anti-inflammatory agents, anti-rheumatic drugs, immunosuppressants, antimetabolites, immunomodulators, antipsoriatics and antidiabetics. A pharmaceutical composition comprising the inhibitory compound of claim 1 and one or more cell adhesion inhibiting compounds. The composition of claim 4, wherein the one or more cell adhesion inhibiting compounds is a VLA-4 inhibitor. 6. A method of preventing, inhibiting or suppressing cellular adhesion in a mammal, comprising the step of administering an effective amount of a pharmaceutical composition of the invention. • ···· ·· ·· · · • ·· · • · · · • · • · • ··· • · · • · • • · • · · · • · · • • · • · · • · • ······ ·· ···· · · · · · year 2, this mammal. A method according to claim 6, characterized in that it is used to prevent, inhibit or suppress inflammation. A method for treating asthma, allergic rhinitis, multiple sclerosis, atherosclerosis, inflammatory bowel disease, or multiple myeloma in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of claim 1. 9. A method of treating multiple sclerosis in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of claim 1. 10. A method of treating an inflammatory bowel disease in a mammal comprising administering to said mammal a therapeutically effective amount of a compound of claim 1. 11. A pharmaceutical composition comprising an ester of a compound of claim 1. 12. The composition of claim 11 wherein the ester is selected from methanol, ethanol, propanol, butanol, and straight or branched chain alkyl alcohols containing from 1 to 10 carbon atoms. 13. A method of treating, preventing or ameliorating symptoms of asthma comprising administering to a patient suffering from asthma a therapeutically effective amount of a compound of claim 1.